EP3052127A1 - Immunstimulierende hiv-tat-derivierende polypeptide zur verwendung bei der krebsbehandlung - Google Patents

Immunstimulierende hiv-tat-derivierende polypeptide zur verwendung bei der krebsbehandlung

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Publication number
EP3052127A1
EP3052127A1 EP14799572.4A EP14799572A EP3052127A1 EP 3052127 A1 EP3052127 A1 EP 3052127A1 EP 14799572 A EP14799572 A EP 14799572A EP 3052127 A1 EP3052127 A1 EP 3052127A1
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EP
European Patent Office
Prior art keywords
cancer
tumor
derivative polypeptide
tat
tat derivative
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP14799572.4A
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English (en)
French (fr)
Inventor
Joshua Goldberg
Colin BIER
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PIN Pharma Inc
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PIN Pharma Inc
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Publication of EP3052127A1 publication Critical patent/EP3052127A1/de
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/664—Amides of phosphorus acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001152—Transcription factors, e.g. SOX or c-MYC
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16071—Demonstrated in vivo effect

Definitions

  • the present invention relates to the field of immune-based therapeutic agents for cancer.
  • Immune checkpoints represent inhibitory molecules that result in the inhibition of an effective immune response towards cancer which can result in tumor evasion.
  • Immune checkpoint molecules such as the cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death 1 (PD-1 ) along with programmed cell death ligand 1 (PD-L1 ) are believed to be contributing to the immune dysfunction that accompanies cancer progression and their therapeutic blockade has shown clinical benefit.
  • CTLA-4 cytotoxic T-lymphocyte antigen 4
  • PD-1 programmed cell death 1
  • PD-L1 programmed cell death ligand 1
  • the engagement of tumor PD-L1 with PD-1 on infiltrating Cytotoxic T lymphocytes (CTL) is believed to be an important mechanism underlying tumor evasion and immune resistance by inducing T-cell anergy, exhaustion, and programmed cell death. Understanding the manipulation of immune checkpoint molecules during the immune response is an important strategy for designing effective immunotherapies for human cancers.
  • the Human Immunodeficiency Virus (HIV) trans-activator of transcription is a variable RNA binding peptide which increases viral RNA transcription and may initiate apoptosis in T4 cells and macrophages and possibly stimulates the over production of alpha interferon.
  • HIV Human Immunodeficiency Virus
  • LTNP long term non-progressors
  • AIDS Acquired Immunodeficiency Syndrome
  • the Tat protein found in LTNP is capable of trans-activating viral RNA; however, this immunostimulatory Tat does not induce apoptosis in T4 cells or macrophages and is not immunosuppressive.
  • immunostimulatory Tat found in lentiviruses that infect monkey species yet do not result in the development of immunodeficiency and epidemic infection direct monocyte differentiation into dendritic cells (DCs) that stimulate cytotoxic T lymphocyte (CTL) responses.
  • DCs dendritic cells
  • CTL cytotoxic T lymphocyte
  • immunostimulatory Tat may have utility in stimulating an immune response towards human cancers.
  • Cancers and chronic infections are the most prominent examples of common human diseases that respond to immune-based treatments. Although infections were the first diseases to be controlled by immunization, clinical trials in humans have established that an immune response, particularly of the CTL arm of the immune system, could regress some human melanomas and renal cancers.
  • DCs a specific class of antigen-presenting cells (APC) are particularly effective at initiating CTL activity against cancers and other diseases.
  • technologies that target and activate DCs have yielded some early successes against human cervical pre- malignancies caused by infection with Human Papilloma Virus (HPV) and human lung cancer.
  • HPV Human Papilloma Virus
  • agents that provoke a CTL response against cancer potentially are accompanied by few side effects, owing to the great specificity of the immune response.
  • Antigen targeting for the induction of a CTL response is a challenge, insofar as natural processing requires that the antigen enter the cytoplasm of the cell in order to bind to the immune system's major histocompatibility complex (MHC) Class I antigen, a prerequisite to CTL activation because the ligand for activating the T cell receptor on CTLs is a complex of antigen and MHC Class I.
  • MHC major histocompatibility complex
  • HSP heat shock proteins
  • a trans-activator of transcription (Tat) derivative polypeptide having an amino acid sequence comprising, in the following order: (i) a transcription factor (TF) domain sequence from a human immunodeficiency virus (HIV) or a simian immunodeficiency virus (SIV) Tat protein, (ii) a cysteine-rich domain sequence from SIV, HIV, or a defensin, and (iii) a C-terminal domain sequence from a HIV or SIV Tat protein.
  • TF transcription factor
  • HAV human immunodeficiency virus
  • SIV simian immunodeficiency virus
  • composition comprising a Tat derivative polypeptide disclosed herein.
  • the HIV is HIV-1 or HIV-2.
  • the HIV-1 Tat is from a long-term non-progressor.
  • the SIV is from a host selected from Table 2.
  • the defensin is an a-defensin or a ⁇ -defensin.
  • the Tat derivative polypeptide further comprises an arginine-rich domain from HIV-1 or HIV-2 Tat.
  • At least one of the amino acids in the TF domain is deleted or substituted with an alanine, an aspartic acid, a glutamic acid, a glycine, a lysine, a glutamine, an arginine, a serine, or a threonine.
  • the at least one substituted amino acid is a proline.
  • the TF domain comprises an amino acid sequence of one of SEQ ID NOs:96-123.
  • the cysteine-rich domain comprises an amino acid sequence of one of SEQ ID NOs: 124-132.
  • the C-terminal domain comprises an amino acid sequence of one of SEQ ID NOs: 133-150.
  • the Tat derivative polypeptide has greater than 85% sequence identity to one of SEQ ID NOs 5-95. In another embodiment, the Tat derivative polypeptide is not one of SEQ ID NOs:2, 3, or 4.
  • Also disclosed herein is a method of treating cancer comprising administering a therapeutically effective amount of a Tat derivative polypeptide or pharmaceutical composition disclosed herein to a subject in need thereof; and causing cessation of growth of the cancer or regression of the cancer in the subject.
  • Also disclosed herein is the use of a therapeutically effective amount of a Tat derivative polypeptide or a pharmaceutical composition to treat cancer in a subject in need thereof, thereby causing cessation of growth of the cancer or regression of the cancer in the subject.
  • Also disclosed herein is a method of reducing tumor burden in a subject with cancer, the method comprising administering a therapeutically effective amount of a Tat derivative polypeptide or a pharmaceutical composition disclosed herein, to a subject in need thereof; and causing regression of the cancer in the subject.
  • Also disclosed herein is the use of a therapeutically effective amount of a Tat derivative polypeptide or a pharmaceutical composition to treat reduce tumor burden in a subject with cancer, thereby causing regression of the cancer in the subject.
  • Also disclosed herein is a method of inhibiting the suppression of an anti-tumor immune response in a subject with cancer, the method comprising administering a therapeutically effective amount of a Tat derivative polypeptide or a pharmaceutical composition disclosed herein to the subject; wherein the administration results in reduction or inhibition of growth of the cancer or in regression of the cancer in the subject.
  • Also disclosed herein is the use of a therapeutically effective amount of a Tat derivative polypeptide or a pharmaceutical composition to inhibit the suppression of an antitumor immune response in a subject with cancer, wherein administration of the Tat derivative polypeptide results in reduction or inhibition of growth of the cancer or in regression of the cancer in the subject.
  • Also disclosed herein is a method of treating a PD-L1 -expressing tumor in a subject with cancer, the method comprising administering a therapeutically effective amount of a Tat derivative polypeptide or a pharmaceutical composition disclosed herein; wherein the administration results in reduction or inhibition of growth of the cancer or in regression of the cancer in the subject.
  • Also disclosed herein is the use of a therapeutically effective amount of a Tat derivative polypeptide or a pharmaceutical composition to treat a PD-L1 -expressing tumor in a subject with cancer, wherein administration of the Tat derivative polypeptide results in reduction or inhibition of growth of the cancer or in regression of the cancer in the subject.
  • the Tat derivative polypeptide has greater than 85% sequence identity to one of SEQ ID NOs 5-95.
  • the Tat derivative polypeptide is administered in a plurality of doses.
  • the administration comprises a repetitive administration cycle wherein each cycle comprises administering a plurality of doses of the Tat derivative polypeptide in a defined time period followed by a rest period and wherein the cycle is repeated a plurality of times.
  • the administration comprises a repetitive administration cycle wherein each cycle comprises administering a plurality of doses of the Tat derivative polypeptide in a defined time period followed by a administration of one or a plurality of doses of a therapeutic agent in a defined time period and wherein the cycle is repeated a plurality of times.
  • the therapeutic agent is cyclophosphamide.
  • the cancer is adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal-cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, bronchial adenomas/carcinoids, carcinoid tumor, cervical cancer, chronic myeloproliferative disorders, colon cancer, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors, eye cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic tumor, glioma, gastric carcinoid, head and neck cancer, heart cancer, hepatocellular cancer, Hodgkin's lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi sarcoma, kidney cancer
  • At least one pre-treatment tumor from the subject contains at least 5% PD-L1 -expressing cells, between 5% and 20% PD-L1 - expressing cells, between 5% and 15% PD-L1 -expressing cells, or between 5% and 10% PD-L1 -expressing cells.
  • FIG. 1 depicts stimulation of human monocytes with Tat derivatives.
  • FIG. 2 depicts a dose-response curve of stimulation of human monocytes with Tat derivatives.
  • FIGs. 3A and 3B depict the effect of therapy with Tat derivatives on 4T1 tumor growth in vitro.
  • BALB/c mice injected with 1 x10 4 4T1 tumor cells were treated with Nani-P1 or Nani-P2 (400 ng, subcutaneous [SC]) (FIG. 3A) or Nani-P3 (400 ng or 2 SC) (FIG. 3B) on days 0, 7, 14 and 21 after injection of tumor cells.
  • the control group was treated with PBS. Data represents mean tumor volume; bars ⁇ SE.
  • Each group contained 10 mice. From day 15, the differences between the control group and groups treated with Nani-P1 or Nani-P2 were significant (p ⁇ 0.05**). The differences between control and Nani-P2 or Nani- P2 was highly significant starting at day 22 (p ⁇ 0.01**). There was no difference between Nani-P3 (either dose) and controls.
  • FIG. 4 depicts a dose response curve for the effects of purified Nani-P2 on 4T1 breast tumor growth in vivo.
  • the fourth, control group was injected in the left flank with PBS.
  • Data represent mean tumor volume.
  • the differences between the control group and 0.4 ng dose was significant (p ⁇ 0.5*), and the difference between control and 4 ng or 40 ng Nani-P2 treated groups was highly significant (p ⁇ 0.1 **, p ⁇ 0.01 **).
  • FIGs. 5A and 5B depict a Kaplan-Meier survival curve of Nani-P2 treatment of mice bearing 4T1 breast tumors.
  • Mice were injected SC with 1 x10 4 4T1 cells in the mammary pad at day 0.
  • Treatment was started at day 0 with four doses of Nani-P2 (40 ng) administered SC.
  • the treatment group had statistically significant better survival over controls (**) (FIG. 5A).
  • therapy was delayed until day 13, at which time a series of three doses of Nani-P2 (40 ng) were administered weekly either intravenous (IV), SC into the draining lymph nodes, or intratumoral (IT) (FIG. 5B).
  • IV Nani-P2 was highly statistically significant at day 47 (**), while the survival benefit of SC Nani-P2 was also statistically significant (*).
  • FIGs. 6A and 6B depict the anti-tumor activity of Nani-P2 in TS/A and SM1 breast carcinoma models. Mice were implanted SC with 1 x10 5 TS/A breast cancer cells (FIG. 6A) and treated with escalating doses of SC Nani-P2 (0.4, 4, and 40 ng). Even at the lowest dose, the primary anti-cancer difference was highly significant (p ⁇ 0.01 **), while the 40 ng dose was also highly significant (p ⁇ 0.01 ***).
  • FIG. 6B depicts mice implanted SC with 2x10 5 SM1 breast cancer cells and treated SC with Nani-P2 (40 ng) on days 0, 7, 14, and 21 . The difference in primary tumor growth between control and Nani-P2 treated SM1 animals was highly statistically significant (p ⁇ 0.01 ***).
  • FIG. 7 depicts INF- ⁇ production from spleen cells of mice bearing 4T1 breast tumors.
  • BALB/c mice were injected SC with 1 x10 4 4T1 cells.
  • Control animals received weekly injections of PBS, while the Nani-P2 treatment comprised once weekly SC injections (40 ng) initiated at day 0 and continued for 4 weeks.
  • SC injections 40 ng
  • the mice were sacrificed, the spleens harvested and frozen as single cell suspensions until time of assay.
  • Spleen cells (2x10 5 ) and 1x10 4 mitomycin C-treated (50 ⁇ g/ml for 30 min) 4T1 stimulator cells (S) were plated into 96-well plates.
  • IFN- ⁇ concentration was determined using a commercial IFN- ⁇ ELISA kit.
  • IFN- ⁇ production was significantly (p ⁇ 0.05*) higher in cultures of spleen cells from Nani-P2-treated mice under all conditions of in vitro culture. 1 : no restimulation; 2: IL-4 (50 ng/ml)/GM-CSF (100 mg/ml); 3: stimulator cells/IL-4/GM-CSF; 4: stimulator cells only. Addition of in vitro agonists IL-4 and GM-CSF (2 and 3) induced highly significant increases in IFN- ⁇ production (p ⁇ 0.01**).
  • FIGs. 8A and 8B depict regression of established 4T1 breast tumors and inhibition of lung metastasis by Nani-P2 treatment.
  • FIG. 8A two groups of 10 BALB/c mice were injected with 1x10 4 4T1 cells in the mammary pad on day 0. One group was dosed with Nani-P2 (40 ng) weekly for three weeks beginning at day 14. A second group was PBS-treated and used as control. Tumor burden was highly significant by day 22 and remained so throughout the duration of the trial (p ⁇ 0.01 ** ). Mice were sacrificed when tumor diameter reached 15 mm, at which time lung metastases were counted (FIG. 8B). Data represent total lung metastases as quantitated by two observers blinded to the treatment protocol (pO.01 ** ).
  • FIG. 9 depicts 4T1 tumor growth and lung metastasis in BALB/c mice.
  • Two groups of 10 BALB/c mice were implanted subcutaneously (SC) with either 1 *10 4 4T1 cells, mice injected IV with 40 ng Nani-P2 or PBS.
  • SC subcutaneously
  • mice were killed and the lungs and tumor were removed, and tumor nodules were counted by eye.
  • Photographs of the tumors and lungs, which were representative of 10 mice, are shown. Whitish tumor lesions can be observed on the surface of the lungs. Three experiments yielded similar results.
  • FIG. 10 depicts Nani-P2 treatment-induced regression of established 4T1 breast tumors.
  • One of 10 mice underwent a complete remission and remained disease-free over 50 days, at which point the study was terminated.
  • Two groups of 10 BALB/c mice were injected with 1x10 4 4T1 cells in the mammary pad on day 0.
  • One group was dosed with Nani-P2 (40 ng) per mouse IV weekly over three weeks beginning at day 14 and the other group was treated with PBS and served as control.
  • the difference in primary tumor growth between control and Nani-P2-treated groups was highly significant (p ⁇ 0.01 ** ).
  • FIG. 1 1 depicts tumor growth after therapy with repeated doses of Nani-P2 and cyclophosphamide.
  • FIG. 12 depicts the survival benefit of repeated doses of Nani-P2 and cyclophosphamide vs. weekly cyclophosphamide.
  • FIG. 13A-B depicts immunohistochemical (IHC) staining of CD8+ cells in spleen tissue from a mouse with 4T1 mammary carcinoma treated with PBS (Control, FIG. 13A) or Nani-P2 (FIG. 13B).
  • IHC immunohistochemical
  • FIGs. 14A-14D depict IHC staining of primary 4T1 breast tumors for PD-L1 and CD8.
  • FIG. 14A depicts IHC staining with PD-L1 antibodies in a PBS control animal. PD-L1 staining was observed in cells with a morphological resemblance to myeloid-derived suppressor cells, tumor-associated macrophage, as well as tumor-associated dendritic cells and fibroblast.
  • FIG. 14B depicts IHC staining in a Nani-P2 treated mouse.
  • FIG. 14C depicts IHC staining of infiltrating CD8+ cytotoxic lymphocytes (CTL) in a PBS control animal.
  • FIG. 14D depicts IHC staining of CD8+ CTL in a Nani-P2 treated mouse.
  • CTL cytotoxic lymphocytes
  • Tat Human Immunodeficiency Virus
  • Tat derivative polypeptides Tat derivatives
  • PINS Precision Immune Stimulants
  • the HIV-Tat protein can repetitively trigger precursor cells of the innate immune lineage into activated antigen presenting cells (APC). These observations have been confirmed in specific reference to the dendritic cell APC, whose activation initiates rounds of HIV replication even in AI DS. Taken together, these data supported the conclusion that Tat had a counter suppressive activity. It is hypothesized that these observations on Tat could be linked to the epidemiological data on breast cancer through the theory that Tat in HIV-infected individuals was chronically stimulating innate immunity thereby restricting breast cancer progression. [0044] Tat Derivative Polypeptides
  • the HIV Tat protein is a variable RNA binding protein of 86 to 1 10 amino acids in length that is encoded on two separate exons of the HIV genome. Tat is highly conserved among all human lentiviruses and is essential for viral replication. When lentivirus Tat binds to the TAR (trans-activation responsive) RNA region, transcription (conversion of viral RNA to DNA and then to messenger RNA) levels increase significantly.
  • Tat increases viral RNA transcription
  • apoptosis programmed cell death
  • macrophages a key part of the body's immune surveillance system for HIV infection
  • a-interferon is a well established immunosuppressive cytokine
  • Extracellular Tat's presence early in the course of HIV infection could reduce a patient's immune response, giving the virus an advantage over the host.
  • the direct destruction of T4 cells and induction of a-interferon production could help explain the lack of a robust cellular immune response seen in AIDS patients, as well as accounting for the initial profound immunosuppression.
  • the Tat protein (SEQ ID NO: 1 ) includes four distinct domains: (1 ) the transduction (SH3) domain (amino acids 3-19); (2) the cysteine-rich ligand binding domain (amino acids 22-37); (3) the membrane translocation sequence (amino acids 47-57) and (4) a tail portion encoded by the second exon (amino acids 73-101 ).
  • the amino terminal portion of Tat includes a short peptide region from a nuclear transcription factor (TF) typically flanked by proline residues. This region determines, at least in part, how stimulatory or suppressive the Tat polypeptide is for cells of the immune system, particularly innate immune cells such as dendritic cells (DC) and macrophages (antigen-presenting cells or APCs). Consequently, it is predicted that modifications to the TF region can render the polypeptides more active in the therapy of cancer and other chronic diseases.
  • TF nuclear transcription factor
  • HIV-1 Tat SH3 binding domain is identical to the sequence found in another TF protein, hairless ⁇ hr), that had previously been shown to have immunosuppressive properties in mice. Mice carrying the hr mutation develop an immune dysregulation, now most commonly called “the TH 1 to TH2 shift," that is the sine qua non of HIV-infected individuals who are progressing to AIDS. Further analysis established that SH3 binding sequence derived from the hr gene is a nearly invariant feature of Tat isolated from HIV-1 , and a very consistent feature of HIV-2.
  • an immunostimulatory Tat derivative polypeptide for the treatment of cancer comprises at least three regions (domains).
  • the first domain is a derivatized nuclear transcription factor (TF) region of Tat
  • the second domain is a cysteine-rich region
  • the third region is a C-terminal Tat domain.
  • TF derivatized nuclear transcription factor
  • Each of these domains comprises a sequence from a Tat protein from a source including, but not limited to, HIV-1 or HIV-2 infected progressors, long-term non-progressors, long-term survivors, elite controllers, and/or SIV infected non- human primate species.
  • cysteine-rich defensin molecules can be substituted in place for a Tat-derived cysteine-rich domain.
  • the cysteine-rich domain from a retrovirus is combined with a TF domain and C-terminal domain from non- human primate Tat sequence.
  • non-human primate cysteine-rich domain is combined with a TF domain and C-terminal domain from a retrovirus.
  • the sequence comprising a fragment of the region which maintains the immunostimulatory activity of the full length domain.
  • retroviruses are SIV, HIV, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Herpes Simplex Virus 1 , Herpes Simplex Virus 2, or equine infectious anemia virus (EIAV).
  • the retrovirus is a lentivirus such as HIV or SIV.
  • the HIV is HIV-1 or HIV-2.
  • Tat derivative polypeptides comprising an amino acid sequence including a transcription factor (TF) domain, a cysteine-rich domain, and a C- terminal domain in that order, wherein each of the TF domain and the C-terminal domain are from a retrovirus Tat protein, and the cysteine-rich domain is from a retrovirus or a defensin, such as a-defensin or ⁇ -defensin.
  • TF transcription factor
  • cysteine-rich domain is from a retrovirus or a defensin, such as a-defensin or ⁇ -defensin.
  • Exemplary non-limiting Tat derivative polypeptides are presented in Table 1 .
  • the TF region has a C-terminal proline residue and the cysteine-rich region has a C-terminal phenylalanine.
  • the modified Tat polypeptide further comprises an arginine-rich domain from a lentiviral Tat protein. The arginine-rich domain is found within the C-terminal region.
  • the TF domain, cysteine-rich domain, and C-terminal domain sequences are arranged in the Tat derivative polypeptide in that order.
  • one or more amino acids, including but not limited to proline, in the TF domain is deleted or substituted with a conservative amino acid substitution, such as with an alanine, an aspartic acid, a glutamic acid, a glycine, a lysine, a glutamine, an arginine, a serine, or a threonine.
  • a conservative amino acid substitution such as with an alanine, an aspartic acid, a glutamic acid, a glycine, a lysine, a glutamine, an arginine, a serine, or a threonine.
  • the TF domain comprises, consists essentially of, or consists of, an amino acid sequence of one of SEQ ID NOs:96-123.
  • the cysteine-rich domain comprises, consists essentially of, or consists of, an amino acid sequence of one of SEQ ID NOs: 124-132.
  • the cysteine-rich domain comprises, consists essentially of, or consists of, an amino acid sequence of one of SEQ ID NOs:133-150.
  • conservatively modified variants refers to variant peptides which have the same or similar biological activity of the original peptides.
  • conservative amino acid changes may be made, which, although they alter the primary sequence of the protein or peptide, do not alter its function.
  • a conservative variant has at least one amino acid substituted by another amino acid or an amino acid analog that has at least one property similar to that of the original amino acid from an exemplary reference peptide. Examples of properties include, without limitation, similar size, topography, charge, hydrophobicity, hydrophilicity, lipophilicity, covalent-bonding capacity, hydrogen-bonding capacity, a physicochemical property, of the like, or any combination thereof.
  • a conservative substitution can be assessed by a variety of factors, such as, e.g., the physical properties of the amino acid being substituted (Table 3) or how the original amino acid would tolerate a substitution (Table 4).
  • Table 3 the physical properties of the amino acid being substituted
  • Table 4 how the original amino acid would tolerate a substitution
  • the selections of which amino acid can be substituted for another amino acid in a peptide disclosed herein are known to a person of ordinary skill in the art.
  • a conservative variant can function in substantially the same manner as the exemplary reference peptide, and can be substituted for the exemplary reference peptide in any aspect of the present specification.
  • a Tat derivative polypeptide is a peptide disclosed in Table 1.
  • the Tat derivative is not one of SEQ ID NOs. 2, 3 or 4.
  • a Tat derivative polypeptide can also comprise conservative variants of a Tat derivative polypeptide.
  • a conservative variant of a Tat derivative polypeptide is a conservative variant of a Tat derivative polypeptide disclosed herein.
  • a conservative variant of a Tat derivative polypeptide can be, for example, an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to a Tat derivative polypeptide.
  • a conservative variant of a Tat derivative polypeptide can be, for example, an amino acid sequence having at most 50%, 55%, 60%, 65%, 70%, 75%, at most 80%, at most 85%, at most 90%, at most 95%, at most 97%, at most 98%, or at most 99% amino acid sequence identity to a Tat derivative polypeptide.
  • a conservative variant of a Tat derivative polypeptide can be, for example, a Tat derivative polypeptide having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30 or more conservative substitutions in the amino acid sequence of a Tat derivative polypeptide.
  • a conservative variant of a Tat derivative polypeptide can be, for example, an amino acid sequence having at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 20, or at least 25 conservative substitutions in the amino acid sequence of a Tat derivative polypeptide.
  • a conservative variant of a Tat derivative polypeptide can be, for example, an amino acid sequence having at most 1 , at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11 , at most 12, at most 13, at most 14, at most 15, at most 20, at most 25, or at most 30 conservative substitutions in the amino acid sequence of a Tat derivative polypeptide.
  • Modifications include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g.. by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
  • polypeptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties.
  • Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids.
  • the peptides disclosed herein are not limited to products of any of the specific exemplary processes listed herein.
  • amino acid sequences which are substantially the same typically share more than 95% amino acid identity. It is recognized, however, that proteins (and DNA or mRNA encoding such proteins) containing less than the above-described level of identity arising as splice variants or that are modified by conservative amino acid substitutions (or substitution of degenerate codons) are contemplated to be within the scope of the present disclosure.
  • various ways have been devised to align sequences for comparison, e.g., Blosum 62 scoring matrix, as described by Henikoff and Henikoff in Proc. Natl. Acad Sci. USA 89:10915 (1992). Algorithms conveniently employed for this purpose are widely available (see, for example, Needleman and Wunsch in J. Mol. Bio. 48:443 (1970).
  • the present disclosure also provides for biologically active fragments of the Tat derivative polypeptides.
  • biologically active fragment refers to fragments of the Tat derivative polypeptides which have immunostimulatory activity.
  • the peptides disclosed herein can self-associate into multimers, including but not limited to, dimers, trimers, and tetramers, in addition to existing in the monomer form. Multimerization of peptides can occur spontaneously or can be facilitated by subjecting the peptides to conditions conducive to multimerization. These conditions are known to persons of ordinary skill in peptide chemistry.
  • the compositions disclosed herein can include monomers or multimers of the peptides, or a mixture of monomers and multimers.
  • the following expression systems are suitable for use in expressing the disclosed Tat derivatives: mammalian cell expression systems such as, but not limited to, Chinese Hamster Ovary (CHO), COS cells (fibroblast-like cells from African green monkey kidney tissue), bovine cells, murine cells, human embryonic kidney cells, or baby hamster kidney cells; insect cell expression systems such as, but not limited to, Bac-to-Bac expression system, baculovirus expression system, and DES expression systems; yeast expression systems: and E. coli expression systems including, but not limited to, pET, pSUMO and GST expression systems.
  • the Tat derivatives are expressed with a histadine (poly histidine) tag useful for isolation of the polypeptide. Histidine tag purification systems are known to persons of ordinary skill in the art.
  • Therapeutically effective amount is intended to qualify the amount required to achieve a therapeutic effect.
  • therapeutically effective amount is synonymous with “therapeutically effective dose” and when used in reference to treating cancer means the most beneficial dose of a composition disclosed herein necessary to achieve the desired therapeutic effect and includes a dose sufficient to reduce tumor size, inhibit growth of a tumor, or cause regression of a tumor.
  • Immune checkpoints such as cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1 ) expressed on tumor-specific T cells, lead to compromised activation and suppressed effector functions such as proliferation, cytokine secretion, and tumor cell lysis. Specifically modulating these receptors with immune checkpoint inhibitors is a new approach in cancer immunotherapy.
  • CTL-4 cytotoxic T-lymphocyte antigen 4
  • PD-1 programmed death 1
  • PD-1 also known as CD279
  • PD-L1 also known as B7-H1 and CD274
  • PD-L2 also known as B7-DC and CD273
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • the immunosuppressive nature of the tumor microenvironment is helpful to explain the immune dysfunction that accompanies cancer progression.
  • the PD-1/PD-L1 signaling pathway is one emerging model for immune evasion at the tumor site and represents an important checkpoint and barrier for an effective immune response.
  • the presence of PD-L1 in the tumor site is considered to facilitate immune evasion as a result of an active tumor-mediated process for reprogramming host cells present in the tumor microenvironment.
  • the engagement of PD-L1 with its PD-1 receptor on the surface infiltrating T-cells may induce their programmed cell death, anergy, and exhaustion.
  • Induction of PD-L1 in the tumor microenvironment may serve as a "molecular shield" to protect the tumor from a cell-mediated immune response.
  • the refractory state of cancers to immunotherapeutics may be a consequence of immunosuppression that accompanies disease progression in established cancers.
  • the Tat derivative polypeptides disclosed herein elicit antitumor immune responses by triggering monocyte-derived dendritic cells to stimulate the CD8+ CTL and override PD-L1 immunosuppression.
  • the PD-1/PD-L1 immunosuppressive signaling pathway may provide a potential mechanism by which breast tumors evade host tumor immunity and therefore Tat derivative polypeptides can impact solid tumor progression by induction of tumor infiltrating CD8+ CTLs in the face of PD-L1 immunosuppression.
  • Modulating of signaling through PD-L1 , thereby preventing PD-L1 from sending a negative co-stimulatory signal to T-cells is likely to enhance immunity in response to infection (e.g., acute and chronic) and tumor immunity.
  • the Tat derivative polypeptides disclosed herein may be combined with antagonists of other components of PD-1 :PD-L1 signaling, for example, antagonist anti-PD-1 and anti-PD-L2 antibodies.
  • agents that modulate immune checkpoints that can be used for immunotherapeutic treatment regimens for cancer in combination with the disclosed Tat derivative polypeptides include, but are not limited to, CTLA-4, PD-1 , PD-L1 , PD-L2, B7-H3, B7-H4, LAG-3, TIM-3, and GITR, and their respective ligands.
  • the disclosed Tat derivatives are immune-stimulating polypeptides which are useful in many types of cancers.
  • the Tat derivatives are useful in treating a type of cancer including, but not limited to, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal-cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, bronchial adenomas/carcinoids, carcinoid tumor, cervical cancer, chronic myeloproliferative disorders, colon cancer, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors, eye cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic tumor, glioma, gastric carcinoid, head and neck cancer, heart cancer, hepato
  • GIST
  • the cancer is breast cancer. In yet another embodiment, the cancer is ovarian cancer. In yet another embodiment, the cancer is prostate cancer. In yet another embodiment, the cancer is lung cancer. In yet another embodiment, the cancer is malignant melanoma.
  • the disclosed Tat derivatives are countersuppressive agents with "stand alone" efficacy in cancer, these observations moreover support the prospect that the Tat derivatives can synergize with other countersuppressive anti-cancer therapeutics currently in clinical development that may have a restricted effect in the face of advanced tumor burden and accompanying severe immunosuppression.
  • a subject is selected for treatment with a Tat derivative polypeptide based on expression of PD-L1 in their tumor tissue.
  • the tumor tissue is evaluated for PD-L1 expression before the subject is treated with any cancer therapy.
  • the tumor tissue is evaluated for PD-L1 expression before the subject is treated with a Tat derivative polypeptide disclosed herein.
  • Expression of PD-L1 may be determined by an immunological analysis of tumor tissue such as, but not limited to, immunohistochemistry, immunoassay (ELISA, ELISPOT, radioimmunoassay), protein microarrays, flow cytometry, quantitative immunofluoresence, and surface plasmon resonance.
  • immunological assays such as quantitative polymerase chain reaction (qPCR), and determination of messenger RNA can also be used.
  • a patient is selected for treatment with the Tat derivative polypeptide if the pre-treatment tumor contains more than 5% PD-L1 -expressing cells, more than 6% PD-L1 -expressing cells, more than 7% PD-L1 -expressing cells, more than 8% PD-L1 -expressing cells, more than 9% PD-L1 -expressing cells, more than 10% PD- L1-expressing cells, more than 1 1 % PD-L1-expressing cells, more than 12% PD-L1- expressing cells, more than 13% PD-L1 -expressing cells, more than 14% PD-L1 -expressing cells, more than 16% PD-L1 -expressing cells, more than 18% PD-L1 -expressing cells, or more than 20% PD-L1 -expressing cells.
  • the present disclosure is also directed to pharmaceutical compositions comprising the above-described Tat derivative polypeptides.
  • Dosages and desired drug concentrations of the disclosed pharmaceutical compositions may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mardenti, J. and Chappell, W. "The use of interspecies scaling in toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi et al, Eds., Pergamon Press, New York 1989, pp. 42-96. In one embodiment, the disease is present. In another embodiment, the life of a cell or an individual is prolonged due to the methods described herein.
  • Tat derivative polypeptides can be formulated without undue experimentation for administration to a mammal, including humans, as appropriate for the particular application. Additionally, proper dosages of the compositions can be determined without undue experimentation using standard dose-response protocols.
  • compositions designed for oral, nasal, lingual, sublingual, buccal, intrabuccal, intravenous, subcutaneous, intramuscular and pulmonary administration can be made without undue experimentation by means well known in the art, for example with an inert diluent or with an pharmaceutically acceptable carrier.
  • the pharmaceutical compositions may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, solutions, syrups, and the like.
  • a "pharmaceutically acceptable carrier” means any of the standard pharmaceutical carriers.
  • suitable carriers are well known in the art and may include but are not limited to any of the standard pharmaceutical carriers like phosphate buffered saline solutions, phosphate buffered saline containing polysorbate 80, water, emulsions such as oil/water emulsion, and various types of wetting agents.
  • Other carriers may also include sterile solutions, tablets, coated tablets, and capsules.
  • Such carriers contain excipients like starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients.
  • Compositions comprising such carriers are formulated by well known conventional methods.
  • the Tat derivative polypeptide compositions can easily be administered parenterally such as for example, by intravenous, intramuscular, intrathecal, or subcutaneous injection.
  • Parenteral administration can be accomplished by incorporating the compounds into a solution or suspension.
  • solutions or suspensions may also include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents.
  • Parenteral formulations may also include antibacterial agents such as for example, benzyl alcohol or methyl parabens, antioxidants such as for example, ascorbic acid or sodium bisulfite and chelating agents such as EDTA.
  • Transdermal administration includes percutaneous absorption of the composition through the skin.
  • Transdermal formulations include patches, iontophoresis devices, ointments, creams, gels, salves and the like.
  • the composition may include various materials which modify the physical form of a solid or liquid dosage unit.
  • the composition may include materials that form a coating shell around the active ingredients.
  • the materials which form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents.
  • the active ingredients may be encased in a gelatin capsule or cachet.
  • the Tat derivative polypeptide compositions of the present disclosure may be administered in a therapeutically effective amount, according to an appropriate dosing regimen.
  • the exact amount required may vary from subject to subject, depending on the subject's species, age and general condition, the severity of the infection, the particular agent(s) and the mode of administration.
  • about 0.001 mg/kg to about 50 mg/kg, of the composition based on the subject's body weight is administered, one or more times a day, to obtain the desired therapeutic effect.
  • about 1 mg/kg to about 25 mg/kg, of the composition based on the subject's body weight is administered, one or more times a day, to obtain the desired therapeutic effect.
  • the total daily dosage of the compositions will be determined by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient or subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient or subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed, and other factors well known in the medical arts.
  • compositions may also be employed in combination therapies. That is, the compositions presently disclosed can be administered concurrently with, prior to, or subsequent to, one or more other desired compositions, therapeutics, treatments or medical procedures.
  • the particular combination of therapies administered will be determined by the attending physician and will take into account compatibility of the treatments and the desired therapeutic effect to be achieved. It will be appreciated that therapeutically active agents utilized in combination may be administered together in a single composition, treatment or procedure, or alternatively may be administered separately.
  • repetitive, or frequent, dosing of the disclosed Tat derivatives is contemplated that could run ahead of tachyphylaxis, as well as reverse the immunosuppressive tide established during cancer progression. Frequent dosing is one procedure used for example in allergy therapy that can support immunological tolerance to an agent. Once the Tat derivative can be used to regain immunoreactivity to a tumor, then other immunotherapeutics that have lost benefit due to advanced disease could potentially regain efficacy. In a second protocol, chemotherapeutic regimens are used that could release a shower of tumor antigens in alternation with Tat derivative immunotherapy. As advanced stage human cancers are often multiply drug resistant, radiotherapy could be a practical alternative in human trials.
  • the number of repeated doses of the Tat derivative polypeptides can be established by the medical professional based on the response of the patient to the doses.
  • the Tat derivative polypeptides is administered once every three days for 3 doses in a ten day period. This administration scheme is then repeated for a plurality of cycles.
  • the present disclosure envisions a variety of different administration schemes wherein the Tat derivative polypeptides is administered multiple times within a selected time frame and then the administration scheme is repeated for a plurality of cycles.
  • administration of the Tat derivative polypeptides can be alternated with administration of one or more other anti-cancer, immunomodulatory, or immunosuppressive agents.
  • the immunosuppressive agent is cyclophosphamide.
  • treatment with the Tat derivative polypeptides can be combined with other cancer therapies such as surgery, radiation therapy, or chemotherapy.
  • Chemotherapeutic agents include alkylating agents such nitrogen mustards, nitrosoureas, tetrazines, aziridines, cisplatins, and derivatives; anti-metabolites such as anti-folates, fluoropyrimidines, deoxynucleoside analogues, and thiopurines; antimicrotubule agents such as vinca alkaloids and taxanes; topoisomerase inhibitors such as camptothecin, irinotecan, topotecan, novobiocin, merbarone, and aclarubicin; cytotoxic antibiotics such as anthracyclines, actinomycin, bleomycin, plicamycin, and mitomycin.
  • the Tat derivatives were equally as effective as when given at the time of tumor implantation when assessed by primary tumor growth, survival, and reduction in metastatic lung burden when compared to PBS-treated controls.
  • Synthetic Tat derivatives are immunostimulatory to APCs, have substantial activity against primary as well as established cancers in three widely-used murine mammary carcinoma models.
  • one of the derivatives, Nani-P2 produced a dose- and route-dependant impact on primary tumor growth, lung metastasis formation, and survival in the aggressive Her2(-) 4T1 breast cancer model. Decreased lung metastases correlate with improved survival, because lung metastasis is the leading cause of mortality in advanced breast cancer.
  • mice bearing established 4T1 breast tumors treated intravenously with Nani-P2 protein had highly significant tumor growth inhibition and survival benefits that extended out at least 36 days past the last dosing.
  • Tat derivatives activate the INF- ⁇ arm of the anticancer T cell immune response (FIG. 5).
  • Baseline levels of INF- ⁇ secreted by splenocytes from mice treated with Nani-P2 are 8-fold higher than that from control mice treated with PBS.
  • IFN- ⁇ secretion in response to Tat derivative treatment in vivo could be additionally augmented (up to 53x) in vitro by innate immune agonists GM-CSF and IL-4, while splenocytes from control mice remain suppressed even after attempts to co-stimulate with high-dose GM-CSF and/or IL4.
  • a more immunogenic breast cancer model (SM1 ) and/or a breast tumor with an immunodominant epitope (TS/A) have a relatively high regression rate after Tat derivative therapy, while the "non-immunogenic" 4T1 model is more refractory.
  • This is consistent with a model that immune suppression is a dominant factor in breast cancer progression, and in fact may be contributory to breast cancer invasiveness.
  • This model is supported by the observation that 4T1 expresses several common breast cancer antigens, including lactadherin and androgen binding protein, at high levels against which the immune response is apparently fully suppressed absent Tat derivative-induced countersuppression.
  • Tat derivative Nani-P2
  • ISS immunostimulatory sequence
  • TLR toll-like receptor
  • LPS lipopolysaccharide
  • mice Female BALB/c mice 6 to 8 weeks old were purchased from the Jackson Laboratory (Bar Harbor, NE). Mice were acclimated for at least 1 week before use. Mice were kept in pathogen-free conditions at the Animal Maintenance Facility of the Columbia University of Medical Center and all experiments were approved by the Institutional Animal Care and Use Committee of Columbia University of Medical Center.
  • mice were injected (SC) with 1x10 4 4T1 , 1x10 5 TS/A or 2 x10 5 SM1 cells, respectively, in the left mammary pad on day 0. Immunotherapy was performed by directly injecting a Tat derivative into the right flank at 0, 7, 12, and 17 days after establishment of tumors. The control group received PBS injection.
  • mice when all of the mice had an established measurable tumor (3-5 mm diameter at 14 days after tumor injection), the animals were randomly assigned to various treatment groups as indicated. Tumor burden (tumor volume) was measured and recorded three times weekly. Animals were sacrificed when tumors reached a volume of 15 mm in diameter and the tumors harvested and weighed.
  • ELISA analysis of IFN- ⁇ production by immune spleen cells Splenocyte secretion of IFN- ⁇ was assessed by an OptEIATM ELISA kit (BD Biosciences). Briefly, spleen cells (1x10 5 /well) from 4T1 tumor-bearing mice were cultured with or without 5x10 3 /well mitomycin C (50 ⁇ g/ml)-treated 4T1 cells (used to provide tumor antigens) in DMEM at a 20:1 E:T (effector:tumor) ratio with IL-2 (50 ng/mL) and GM-CSF (100 ng/ml) in 96-well plates.
  • IL-2 50 ng/mL
  • GM-CSF 100 ng/ml
  • IFN- ⁇ was measured in cell-free supernatants of duplicate wells by ELISA according to the manufacturer's instructions. Tumor-specific IFN- ⁇ production was calculated by subtracting the background values measured in supernatants of spleen cells cultured with medium alone and optical density (OD) values were converted to pg/ml amounts of IFN- ⁇ using a recombinant IFN- ⁇ standard curve. Stimulation index (SI) was calculated as the ratio of IFN- ⁇ in stimulated versus control cultures.
  • Nani-P1 and Nani-P2 significantly reduced tumor burden when compared to control mice receiving PBS injections alone, with this difference first becoming apparent at 15 days after tumor implantation (FIG. 3A, day 15 p ⁇ 0.05).
  • Nani-P2 was significantly more sustained than Nani-P1 , so that at day 21 (the final dosing), the difference in primary tumor burden between Nani-P2 and Nani-P1 -treated tumors became 18 mm 3 and was highly statistically significant (p ⁇ 0.01 ). This effect persisted throughout the remainder of this trial despite no further therapy.
  • Nani-P2 treatment could extend survival in addition to shrinking primary tumors in mice.
  • treatment protocols using various dosing schedules and routes (SC, IV or IT) of administration of 40 ng Nani-P2 were performed. Cohorts of ten mice per group were followed for length of survival, as assessed by use of the Kaplan-Meier product limit method. As per Columbia University Medical Center Animal Facility regulations, each mouse was euthanized at a mean tumor diameter of approximately 15 mm, or earlier if the mouse became moribund, making one of these two outcomes the defining criteria for fatality.
  • SC treatment was initiated simultaneously to tumor implant.
  • mice The median survival time for control (PBS treated) mice was 30 days and 100% fatality occurred by day 36.
  • Nani-P2 administration 4 doses over 21 days
  • 35% of treated mice were still alive at day 48 (p ⁇ 0.001 , FIG. 5A) at which point all of the mice were sacrificed due to primary tumor burden.
  • the 4T1 murine mammary tumor model was chosen for study because it is an aggressive and rapidly invasive tumor; it is routinely metastatic at fourteen days post-implant by which time it is difficult to treat.
  • TS/A and SM1 were studied (FIG. 6).
  • TS/A primary mammary tumors were approximately as aggressive as 4T1 , reaching a tumor volume of 15 mm at 30 days (FIG. 6A).
  • the TS/A tumors were considerably more responsive to Nani-P2 treatment, with an approximate 50% suppression of growth after treatment with 0.4 ng Nani-P2, and a 40% total remission rate at 30 days.
  • the SM1 mammary carcinoma model (FIG. 6B) is initially less aggressive as a primary tumor, and deaths appear to be through mechanisms other than metastatic disease.
  • SM1 tumors reached a mean volume approximately 33% smaller than either TS/A or 4T1.
  • FIG. 7 To determine whether cytotoxic T-lymphocytes play a role in tumor rejection induced by Nani-P2 therapy, an IFN- ⁇ ELISA assay (FIG. 7) was performed to compare spleen cells of 4T1 tumor-bearing mice treated either without (Control) or with Nani-P2 (FIG. 7). Spleens were removed under sterile conditions and prepared as described elsewhere (duPre' S. et al. Exp. Mol. Path. 85:174-188, 2008). Briefly, spleens were homogenized and splenocytes, as a rich source of systemic cytolytic T cells and APCs, were co-cultured with mitomycin C-treated 4T1 stimulator cells to induce recall immune responses. Control wells were cultured with medium alone.
  • IFN- ⁇ concentrations a standard surrogate for CTL activation, were quantitated by commercial ELISA (BD Biosciences). IFN- ⁇ production was significantly higher (p ⁇ 0.01 **) in cultures of spleen cells taken from Nani-P2-treated BALB/c mice under all conditions of assay.
  • mice treated IV with Nani-P2 had an average of 35.3 (p ⁇ 0.01 **). This corresponded to a less aggressive appearance of primary tumor, as well as lung metastases that were on average much smaller in size (FIG. 8B).
  • Nani-P2 efficacy in the setting of pre-established, aggressive 4T1 breast cancer is clearly and significantly proven by comparing primary tumor burden in intravenously- treated animals (40 ng IV Nani-P2) against control (PBS-treated) animals (at day 18 p ⁇ 0.01 **, FIG. 10).
  • This statistically significant benefit in primary tumor suppression (FIG. 10) remained throughout the duration of the trial lasting 50 days (p ⁇ 0.01 **) even though only three weekly doses of Tat derivative polypeptide were administered between days 14 and 28.
  • one animal underwent a complete remission and remained disease-free at 50 days, at which point the study was terminated, supporting the inference that this animal had been rendered apparently tumor-free.
  • mice Four groups of 10 BALB/c mice were implanted with 1x10 4 4T1 cells SC into the mammary fat pad. Treatment was initiated when tumor diameters reached 4-5 mm, on day 10. Control mice were injected IV with PBS at 3 days intervals, while alternating treatment mice received 3 doses of drug every 3 days in rotating 10 day cycles. Tumor burden (tumor size mm 3 ) was calculated using a standard formula. CY (cyclophosphamide alone) mice were injected IP weekly with 80 mg/kg per mouse beginning on day 10.
  • Cy/Nani-P2 mice were first injected IP with cyclophosphamide (80 mg/kg) at 3 days intervals for three doses starting at day 10 and then injected IV with Nani-P2 (40 ng) at 3 days intervals for three doses in rotation. The cycle of 3 doses of CY followed by 3 doses of Nani-P2 was repeated until day 50.
  • Nani-P2/CY Nani- P2 first followed by cyclophosphamide mice were first injected IV with Nani-P2 (40 ng) at 3 day intervals for 3 doses starting on day 10 and then injected i.p. with cyclophosphamide at 3 day intervals in rotation. The cycle of 3 doses of Nani-P2 followed by 3 doses of CY was repeated until day 50.
  • the Nani-P2 cohort has 3/10 mice in total remission and 9/10 mice in partial remission at day 50 (not shown), while 10/10 cyclophosphamide treated mice were dead by day 42.
  • the spleen is a major lymphoid organ and site where antigen presenting cells display captured tumor associated antigens to stimulate cytotoxic T-cell responses. Tumor specific CTLs will migrate to the site of infection and lyse the target cell.
  • mice Female BALB/c mice were inoculated in the mammary fat pad with syngeneic and highly metastatic 4T1 breast cancer cells to model Stage IV human breast cancer. Nani-P2 immunotherapy was initiated 7 days after tumor cell inoculation. Tumors were assessed by caliper measurements throughout the study and resected on Day 29/30. Immunohistochemical staining (IHC) and CD8 was performed on formalin-fixed, paraffin embedded specimens of resected spleen tissue. [0129] As depicted in FIG. 13, IHC staining reveals increased populations of splenic mouse CD8+ cells following treatment with Tat derivatives (FIG. 13B) versus no treatment (PBS, FIG. 13A)).
  • the refractory state of cancers to immunotherapeutics may be a consequence of immunosuppression that accompanies disease progression in established cancers.
  • PD-L1 programmed cell death receptor- ligand-1
  • CTL cytotoxic T- lymphocytes
  • the Tat derivative polypeptides disclosed herein elicit antitumor immune responses by triggering monocyte-derived dendritic cells to stimulate the CD8+ CTL and override PD-L1 immunosuppression.
  • the PD-1/PD-L1 immunosuppressive signaling pathway may provide a potential mechanism by which 4T1 tumors evade host tumor immunity and therefore Tat derivative polypeptides can impact solid tumor progression by induction of tumor infiltrating CD8+ CTLs in the face of PD-L1 immunosuppression.
  • mice Female BALB/c mice were inoculated in the mammary fat pad with syngeneic and highly metastatic 4T1 breast cancer cells to model Stage IV human breast cancer. Nani-P2 immunotherapy was initiated 7 days after tumor cell inoculation. Tumors were assessed by caliper measurements throughout the study and resected on Day 29/30. Immunohistochemical staining (IHC) for PD-L1 and CD8 was performed on formalin-fixed, paraffin embedded specimens of primary 4T1 tumors.
  • IHC Immunohistochemical staining
  • PD-L1 expression is reduced in animals receiving Nani- P2 treatment (FIG. 14B) versus controls (FIG. 14A).
  • PD-L1 staining was observed in cells with a morphological resemblance to myeloid-derived suppressor cells, tumor-associated macrophage, as well as tumor-associated dendritic cells and fibroblast.
  • PD-L1 reduction is based on in vivo tumor measurement data in Nani-P2 treated vs. control, combined with less PD-L1 staining intensity. Tumor edge containing majority of PD-L1 staining is largely absent in Nani-P2 treated as compared to control.
  • Tumor-infiltrating CD8+ CTLs appear to localize near the tumor edge in Nani-P2 treated mice, where as these CTLs are largely absent in tumor edges of PBS control. Since PD-L1 is a marker associated with disease progression, malignancy, and poor prognosis, the inverse correlation of tumor PD- L1 and CD8+ CTL can be explained based on the antitumor CTL response observed with PIN-2 treatment.

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RU2016112538A (ru) 2017-11-13
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IL244867B (en) 2020-01-30
HK1221910A1 (zh) 2017-06-16
RU2016112538A3 (de) 2018-08-23
US20150098960A1 (en) 2015-04-09
AU2014329393B2 (en) 2020-04-30
US20170274040A1 (en) 2017-09-28
US9663556B2 (en) 2017-05-30
RU2695653C2 (ru) 2019-07-25
AU2014329393A1 (en) 2016-04-28
IL244867A0 (en) 2016-05-31
CA2926221A1 (en) 2015-04-09
WO2015051245A1 (en) 2015-04-09
ZA201602196B (en) 2017-11-29
US20190030117A1 (en) 2019-01-31
JP2016533352A (ja) 2016-10-27

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